Troubleshooting Homogenization and Western Protocols for MeT1 in Mouse Muscle and Brain

An Honors Thesis (HONR 499)

By

Audrey Aldrich

Thesis Advisor

Dr Bartholomew Pederson

Ball State University Muncie IN

May 2014

Expected Date of Graduation

May 2014

c )) Un deg3rad

ampj

egiS

zIt g 0 ) t Abstract rl LI1

The fact that the body transforms the senses sight touch hearing smell and taste from abstract ideas into memory by the tangible movement of metabolites such as glucose and lactate remains a sort of miracle and a mystery However scientists and researchers are quickly beginning to discover the metabolic pathway used to store memories and even recollect them Thus far it has been proven that glucose as in many other pathways is a large and necessary component The processing of senses into memories also largely requires lactate and currently this is the metabolite receiving focus by the scientific community The lactate necessary for memory processing is found in astrocytes and transported to neurons by way of a shuttle system Because of its large size MCT] and MCT2 (monocarboxylate transporters) are needed to move the lactate in and out of the astrocyteneuron This project focuses on a method to demonstrate that the MCT] and MCT2 proteins can be detected in brain tissue and then compare the amounts of these proteins in different mouse types and after varying mentalphysical exercises

Acknowledgements

I would like to thank Dr Bartholomew Pederson for allowing me to work in his lab furthering my knowledge of biochemistry and for advising me on this project Furthermore I would like to thank the CRlSP faculty and staff as well as the Ball State Chemistry department who gave me the opportunity to participate in Research Lastly I would like to thank the Ball State Honors College for pushing me to grow in my education and as an individual

Introduction

Metabolites such as glucose and lactate as well as glycogen (the storage counterpart of glucose) playa large role in regulating energy production throughout the human body Glycogen is also set aside as energy storage mainly in muscle and liver for later use by the body When the blood glucose levels are low (therefore energy or

P age 1

ATP are low) glucagon is secreted by a-cells in the pancreas which then causes glycogen stores to break down and stimulates gluconeogenesis When the blood glucose levels are high the opposite occurs insulin is secreted by ~-cells in the pancreas causing removal of glucose from the blood into storage as lipids or glycogen (Berg et at 20] 2)

The human body however can only handle so many lipids Excess lipid storage results in obesity which in tum may cause diabetes mellitus heart disease and hypertension (Berg et al 2012) Diabetes is defined as a disease in which the liver overproduces glucose while other organs underuse glucose There are two types of diabetes types I and II Type I diabetes results from the autoimmune destruction of ~-cells in the pancreas Therefore insulin cannot be produced and afflicted individuals are insulin dependent meaning they need insulin shots to regulate glucose metabolism Type II diabetes on the other hand is deemed insulin resistant so the body does not respond to insulin (Berg et al 2012)

There are many health dangers associated with diabetes such as loss of eyesight or loss of limbs but potentially as detrimental is hypoglycemia With hypoglycemia comes loss of cognitive function and in severe cases neuronal death Hypoglycemia is caused by low blood glucose levels and while lactate amino acids and ketone bodies are important glucose is the main source of energy for the brain When blood glucose concentration falls below a certain level the brain glucose levels fall quickly because consumption of glucose is occurring faster than glucose can be transported to the brain from other parts of the body (Amaral 2012)

This honors thesis however specifically looks to address the role of lactate in the brains process of memory storage and recollection Thus far research has determ ined that the concentration of extracellular lactate increases within a learning environment and during long term

memory formation Further proving the importance of lactate blockage of monocarboxy late transporters (lactate transporters) causes amnesia in test subjects (Suzuki 2011) The increase in extracellular lactate occurs through the glycogenolysis of intercellular glycogen or through the glycolysis of extracellular glucose which is seen to decrease during spatial learning (Newman and Gold 2011) The process of glycogenolysis occurs in the astrocyte where lactate is produced It is then presumed that lactate is transported into neurons to support further metabolic functions (Newman and Gold) The MCTI and MCT2 proteins focused on in this project represent the order of transportation MCTI is an astrocytic transporter and MCT2 acts as a neuronal transporter of lactate The measurement of lactate transport in and out of the astrocytesneurons can be related to the relative amounts of MCTI and MCT2 proteins When a specific metabolic process is in high demand so are the proteins (Bezzi 2011)

After the MCTI and MCT2 proteins are reliably detected the relative amounts will be compared in three different genotypes of mice as well as the levels (through observation of Western data) after varying physical and mental tests The types of mice used include HO HOG and wild type mice Wild type mice exhibit the nonnal expression of glycogen synthase so nonnal levels of glycogen are present in muscle and liver tissue HO mice on the other hand are genetically engineered to lack glycogen synthase so no glycogen stores can be found in their muscle or brain HOG mice are also engineered to lack glycogen synthase in brain tissue but overproduce glycogen in muscle A few of the tests perfonned on the mice include wire hang rotarod object recognition and passive avoidance The wire hang measures the endurance of the mouse in accordance to the amount of available glycogen in its muscles The rotarod measures motor learning The object recognition test focuses on recognition and the passive avoidance tests measures associative memory

Discovering the role of glucose glycogen and lactate in the metabolic process of memory

Page 12

storage and recollection may influence the treatment of patients suffering from hypoglycemia Alzheimers disease or other diseases which effect memory Thus far some studies have determined that injections of epinephrine glucose and lactate increase memory storage and recollection in mice (Gold and Korol 2012)

Methods

Homo enization

The first step in detennining the relative amounts of lactate transporters prevalent in mouse brain tissue after spatial learning exercises and hypoglycemiahomeostasis is to establish a reliable method for detection This particular set of methods and data focus on optimizing the protocol for preparing tissue homogenates and perfonning Western blots in accordance with detecting MCTI Besides its location in astrocytes MCTI is also located in larger quantities throughout the body so for the purpose of detection mouse muscle tissue was homogenized and used

Tissue homogenization and preparation was tried several different ways The following procedure was proven to yield the best results but still requires modification to ensure the proteins presence First mice were sacrificed using cervical dislocation to reduce the amount of metabolites lost and then leg muscles were harvested The muscles were immediately placed in nitrogen for preservation Further preservation required storage in a -80degC freezer During tissue preparation for homogenization samples were also kept in nitrogen The tissue which was to be homogenized and prepared for future Bradford Assays and Western Blots was weighed out and homogenized using a buffer made up of

1 ml-SOmM Tris pH 7810 mM EDTA2mM EGTA1 OOmM NaF

37111 10 mgmL TLCK 2 ilL 1 M benzamidine SilL 100 mM PMSF 35 ilL BME 1 ilL 10 mgm L leupeptin

The samples were then centrifuged for 5 min at 3600xg at 4degC The supernatant was saved for Bradfords and Westerns Supernatant meant for Westerns was prepared with 5X SDS-PAGE loading buffer containing BME (2shymercaptoethanol) The samples were then boiled for 5 minutes then placed in -20deg C storage

EDTA Ethylenediaminetetraacetic acid EGTA Ethy lene-bis( oxyethy lenenitri 10)tetraacetic acid TLCK Tosyllysine chloromethylketone PMSF Phenylmethylsulfonyl Fluoride

SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)

The Western protocol focused on detecting MCT1 at 40-48 kDa Once again several protocols were used to detect the protein but the following protocol produced the best results Like mentioned before further modifications are necessary to affirm the presence of the MCT1 protein Using Bio-Rad Mini-PROTEAN TGX (cat456-1033) 1000 precast gels wells were loaded with 20lL of protein and the appropriate amounts of 5X buffer and homogenization buffer The gel was run for ~35 min at a voltage of 200 Next the proteins from the gel were transferred onto a nitrocellulose membrane See figure 1 as an example of how the transfer worked

Western

The membrane was then blocked overnight at 4deg C in lXTBSTweenl400 milk Next the membrane was exposed to a 1 200 MCTI dilution in 1XTBS005tween-20500milk for an hour at room temperature To determine the proteins bound by the antibodies blocking peptides were used The membrane was then washed with 1 XTBSTween two times for a few seconds each then three times for ten minutes each The membrane was then exposed to a 1 1 0000 dilution of the secondary anti-goat antibody in lXTBS00500Tween500milk for one hour at room temperature The membrane was washed again with the same protocol as

P a ge I 3

above but with an extra step after the initial wash the membrane was placed in 1XTBS for 5 minutes Lastly before imaging the ECL (Electrochemiluminescence) reagent (Supersignal West stable peroxide solution mixed with Supersignal West pico luminolenhancer solution) was added to the membrane and rocked for 5 minutes After 5 minutes the membrane was imaged (Kodak Image Station 440)

TBS Tert-Butyldimethylsilyl Ether

Antibodies Used Santa Cruz Biotechnology MCT1 (T-19) Scshy14917 lot 01311 goat polyclonal IgG Santa Cruz Biotechnology IgG-HRP Sc-2020 Lot B0513 HRP conjugated Blocking Peptide Santa Crus Biotechnology MCTI (T-19)P Scshy14917 P Lot G 1212 Blocking Peptide

Data

10 9 8 7 5 4 3 2

--40

Figure 1 This is an image of the nitrocellulose membrane stained in Ponceau after the transfer While the lanes look more smeared than normal it is apparent that proteins residing at ~40 kDa are present

Figure 2 This image shows proteins from prepared muscle homogenates The left half shows non-centrifuged homogenates while the right half shows centrifuged homogenates (13000 rpm at room tern perature) Lanes 2 and 7 represent the protein samples prepared by using Santa Cruz reagents 5X loading buffer and then boiled for 3 minutes Lanes 3 and 8 represent the protocol for preparing homogenates portrayed in this paper Lanes 4 and 9 represent proteins of homogenates made using Santa Cruz Reagents Santa Cruz 2X buffer and boiled for 10 minutes Lanes 5 andlO represent proteins of homogenates made using the Santa Cruz reagents Santa Cruz 2X loading buffer BME and boiled for 10 minutes Lane 2 shows the most promise by presenting with three bands one at 40 kOa and two above 70 kOa It is possible the larger proteins exist as tetramers and dimers of the MCTI protein

Figure 3 This image shows a completed Western using different techniques for denaturation of the protein in order to increase

P a g e 14

separation The membrane on the right was treated with blocking peptides Lanes 2 and 7 represent proteins prepared using 2X SOSshyPAGE loading buffer and BME then boiled for 5 minutes Lanes 3 and 8 represent proteins prepared using 2X SOS-PAGE loading buffer and BME then heated to 37deg C for 30 minutes Lanes 4 and 9 represent proteins prepared using 2X SOS-PAGE loading buffer with BME and heated to 70deg C for 10 minutes Lanes 5 and 10 represent proteins prepared using 2X SOSshyPAGE loading buffer with OTT (Oithiothreitol) then boiled for 5 minutes This procedure did not result in convincing detection of the MCTl protein

Results

The Western pictured in figure 2 (lane 3) potentially shows the MCTI protein at ~40 kOa However two other bands also persist darker and more prominent than the band at 40kDa The other bands may in fact be dimers or tetramers of the MCTI protein which did not efficiently break apart during sample preparation In order to further degrade the MCT1 proteins a higher concentration of BME was experimented with along with different heating methods after the addition of BME One sample was even prepared with OTT instead of BME As figure 2 shows the higher concentrations of BME and the sample of OTT did not result in a single dark band at ~40 kOa To ensure the bands present were dimers or tetramers of the MCT1 protein a blocking peptide was added to a second membrane The bands seen in figure 3 did not disappear with the blocking peptide and therefore it is assumed the bands are not associated with the MCTI protein Another technique performed to isolate the MCTl protein from the interfering proteins included different centrifugation styles However as shown in figure 3 the centrifugation after homogenization completely eliminated any traces of the MCT1 protein

Discussion

Thus far troubleshooting with blocking peptides differing concentrations of BME the addition of OTT changes in heating temperatures and time the use of milk vs BSA

(Bovine Serum Albumin) and centrifugation techniques has not yielded viable results for determining the presence of MCTI in muscle Further troubleshooting includes the use of urea a positive control or a more sensitive ECL reagent Urea like SDS causes the further denaturation of proteins and a sample with overexpressed MCT 1 to act as a positive control to ensure the functionality of the antibody Possible changes in technique include longer exposure time for the membrane or the use of a tissue other than muscle As a last resort a new MCT 1 antibody should be tried Other research has proved the location and existence of the MCTI protein in muscle therefore the possibility stands that the antibody used in these experiments was faulty

Conclusions

Overall the troubleshooting performed during these experiments allowed for the betterment of the Homogenization preparation protocol and the Western protocol The troubleshooting also eliminated many procedures For example strong centrifugation after the addition of SDS buffer with BME results in the loss of the MCTI protein into the pellet Another a stronger concentration of BME or even switching to DTT does not break apart the tetramers and dimers of the MCT 1 protein Hopefully urea will do what SDS did not by breaking apart the tetramers and dimers to yield a protein band at ~40kDa and cross checked with the blocking peptide If the urea does not work the next step is to use a positive antibody as a positive control to confirm that the current antibody is attaching to the right protein Like mentioned previously a new antibody may have to be selected and the process of troubleshooting may have to start back at square one

Eventually the MCTI protein will be confirmed at ~40 kDa and mark the next step to uncovering the secret behind memory storage and recollection Further experiments will be performed comparing the level of the MCT1 proteins between differing mouse genotypes and the existing levels after differing behavioral activities The resulting comparisons and

P age I 5

analysis will allow insight into what occurs with the MCTI protein and lactate levels in the brain during hypoglycemia Medical fields dealing with diabetes Alzheimers disease or other metabolic deficiencies affecting cognitive function hope to benefit from this research and similar research in the near future

References

Amaral A 1 (2012) Effects of Hypoglycemia on Neuronal Metabolism in the Adult Brain Role of Alternative Substrates to Glucose Inherit Metab Dis Doi 101007510545-012-9553-3

Berg J M Tymoczko J L amp Stryer L (2012) BiochemistryNew York W H Freeman amp Company

Bezzi P amp Voltera A (2011) Astrocytes Pwering Memeroy Cell 144 Doi 101016jceI1201102027

Gold P E amp Korol D L (2012) Making Memories Matter Frontiers in Integrative Neuroscience 6(166) Doi 103389frint201200116

Newman L A Korol D L Gold P E (2011) Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing PLos ONE 6(12 Doi 10 13711joumalpone0028427

Suzuki A et al (2011) Astrocyte-Neuron Lactate Transport Is Required for Long Term Memory Formation Cell 144 810-823 Doi 101016jceI1201102018

Completion Report httpsllwwwcitiprogramorgmemberslearnersllicrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with Mice in Research Settings Curriculum Completion Report Printed on 5152013

Leamer Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Working with Mice in Research

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343726)

Elective Modules Date

Completed Score Introduction to Working with Mice in Research Settings 051513 no quiz

Research Mandates and Occupational Health Issues 051513 313 (1 00 )

Alternatives Searches Humane Standards Housing and Acclimation and Quarantine

0511513 212 (100)

Detecting Pain and Distress Genetics and Biological Features

0511513 313 (100)

Injections Blood Collection and Antibody Production 0511513 212 (10000)

Surgery Supportive Care and Monitoring Euthanasia and References

051513 415 (80)

For this Completion Report to be valid the learner Ilisted above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

Return

1 of 1 51512013 5 18 PM

c )) Un deg3rad

ampj

egiS

zIt g 0 ) t Abstract rl LI1

The fact that the body transforms the senses sight touch hearing smell and taste from abstract ideas into memory by the tangible movement of metabolites such as glucose and lactate remains a sort of miracle and a mystery However scientists and researchers are quickly beginning to discover the metabolic pathway used to store memories and even recollect them Thus far it has been proven that glucose as in many other pathways is a large and necessary component The processing of senses into memories also largely requires lactate and currently this is the metabolite receiving focus by the scientific community The lactate necessary for memory processing is found in astrocytes and transported to neurons by way of a shuttle system Because of its large size MCT] and MCT2 (monocarboxylate transporters) are needed to move the lactate in and out of the astrocyteneuron This project focuses on a method to demonstrate that the MCT] and MCT2 proteins can be detected in brain tissue and then compare the amounts of these proteins in different mouse types and after varying mentalphysical exercises

Acknowledgements

I would like to thank Dr Bartholomew Pederson for allowing me to work in his lab furthering my knowledge of biochemistry and for advising me on this project Furthermore I would like to thank the CRlSP faculty and staff as well as the Ball State Chemistry department who gave me the opportunity to participate in Research Lastly I would like to thank the Ball State Honors College for pushing me to grow in my education and as an individual

Introduction

Metabolites such as glucose and lactate as well as glycogen (the storage counterpart of glucose) playa large role in regulating energy production throughout the human body Glycogen is also set aside as energy storage mainly in muscle and liver for later use by the body When the blood glucose levels are low (therefore energy or

P age 1

ATP are low) glucagon is secreted by a-cells in the pancreas which then causes glycogen stores to break down and stimulates gluconeogenesis When the blood glucose levels are high the opposite occurs insulin is secreted by ~-cells in the pancreas causing removal of glucose from the blood into storage as lipids or glycogen (Berg et at 20] 2)

The human body however can only handle so many lipids Excess lipid storage results in obesity which in tum may cause diabetes mellitus heart disease and hypertension (Berg et al 2012) Diabetes is defined as a disease in which the liver overproduces glucose while other organs underuse glucose There are two types of diabetes types I and II Type I diabetes results from the autoimmune destruction of ~-cells in the pancreas Therefore insulin cannot be produced and afflicted individuals are insulin dependent meaning they need insulin shots to regulate glucose metabolism Type II diabetes on the other hand is deemed insulin resistant so the body does not respond to insulin (Berg et al 2012)

There are many health dangers associated with diabetes such as loss of eyesight or loss of limbs but potentially as detrimental is hypoglycemia With hypoglycemia comes loss of cognitive function and in severe cases neuronal death Hypoglycemia is caused by low blood glucose levels and while lactate amino acids and ketone bodies are important glucose is the main source of energy for the brain When blood glucose concentration falls below a certain level the brain glucose levels fall quickly because consumption of glucose is occurring faster than glucose can be transported to the brain from other parts of the body (Amaral 2012)

This honors thesis however specifically looks to address the role of lactate in the brains process of memory storage and recollection Thus far research has determ ined that the concentration of extracellular lactate increases within a learning environment and during long term

memory formation Further proving the importance of lactate blockage of monocarboxy late transporters (lactate transporters) causes amnesia in test subjects (Suzuki 2011) The increase in extracellular lactate occurs through the glycogenolysis of intercellular glycogen or through the glycolysis of extracellular glucose which is seen to decrease during spatial learning (Newman and Gold 2011) The process of glycogenolysis occurs in the astrocyte where lactate is produced It is then presumed that lactate is transported into neurons to support further metabolic functions (Newman and Gold) The MCTI and MCT2 proteins focused on in this project represent the order of transportation MCTI is an astrocytic transporter and MCT2 acts as a neuronal transporter of lactate The measurement of lactate transport in and out of the astrocytesneurons can be related to the relative amounts of MCTI and MCT2 proteins When a specific metabolic process is in high demand so are the proteins (Bezzi 2011)

After the MCTI and MCT2 proteins are reliably detected the relative amounts will be compared in three different genotypes of mice as well as the levels (through observation of Western data) after varying physical and mental tests The types of mice used include HO HOG and wild type mice Wild type mice exhibit the nonnal expression of glycogen synthase so nonnal levels of glycogen are present in muscle and liver tissue HO mice on the other hand are genetically engineered to lack glycogen synthase so no glycogen stores can be found in their muscle or brain HOG mice are also engineered to lack glycogen synthase in brain tissue but overproduce glycogen in muscle A few of the tests perfonned on the mice include wire hang rotarod object recognition and passive avoidance The wire hang measures the endurance of the mouse in accordance to the amount of available glycogen in its muscles The rotarod measures motor learning The object recognition test focuses on recognition and the passive avoidance tests measures associative memory

Discovering the role of glucose glycogen and lactate in the metabolic process of memory

Page 12

storage and recollection may influence the treatment of patients suffering from hypoglycemia Alzheimers disease or other diseases which effect memory Thus far some studies have determined that injections of epinephrine glucose and lactate increase memory storage and recollection in mice (Gold and Korol 2012)

Methods

Homo enization

The first step in detennining the relative amounts of lactate transporters prevalent in mouse brain tissue after spatial learning exercises and hypoglycemiahomeostasis is to establish a reliable method for detection This particular set of methods and data focus on optimizing the protocol for preparing tissue homogenates and perfonning Western blots in accordance with detecting MCTI Besides its location in astrocytes MCTI is also located in larger quantities throughout the body so for the purpose of detection mouse muscle tissue was homogenized and used

Tissue homogenization and preparation was tried several different ways The following procedure was proven to yield the best results but still requires modification to ensure the proteins presence First mice were sacrificed using cervical dislocation to reduce the amount of metabolites lost and then leg muscles were harvested The muscles were immediately placed in nitrogen for preservation Further preservation required storage in a -80degC freezer During tissue preparation for homogenization samples were also kept in nitrogen The tissue which was to be homogenized and prepared for future Bradford Assays and Western Blots was weighed out and homogenized using a buffer made up of

1 ml-SOmM Tris pH 7810 mM EDTA2mM EGTA1 OOmM NaF

37111 10 mgmL TLCK 2 ilL 1 M benzamidine SilL 100 mM PMSF 35 ilL BME 1 ilL 10 mgm L leupeptin

The samples were then centrifuged for 5 min at 3600xg at 4degC The supernatant was saved for Bradfords and Westerns Supernatant meant for Westerns was prepared with 5X SDS-PAGE loading buffer containing BME (2shymercaptoethanol) The samples were then boiled for 5 minutes then placed in -20deg C storage

EDTA Ethylenediaminetetraacetic acid EGTA Ethy lene-bis( oxyethy lenenitri 10)tetraacetic acid TLCK Tosyllysine chloromethylketone PMSF Phenylmethylsulfonyl Fluoride

SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)

The Western protocol focused on detecting MCT1 at 40-48 kDa Once again several protocols were used to detect the protein but the following protocol produced the best results Like mentioned before further modifications are necessary to affirm the presence of the MCT1 protein Using Bio-Rad Mini-PROTEAN TGX (cat456-1033) 1000 precast gels wells were loaded with 20lL of protein and the appropriate amounts of 5X buffer and homogenization buffer The gel was run for ~35 min at a voltage of 200 Next the proteins from the gel were transferred onto a nitrocellulose membrane See figure 1 as an example of how the transfer worked

Western

The membrane was then blocked overnight at 4deg C in lXTBSTweenl400 milk Next the membrane was exposed to a 1 200 MCTI dilution in 1XTBS005tween-20500milk for an hour at room temperature To determine the proteins bound by the antibodies blocking peptides were used The membrane was then washed with 1 XTBSTween two times for a few seconds each then three times for ten minutes each The membrane was then exposed to a 1 1 0000 dilution of the secondary anti-goat antibody in lXTBS00500Tween500milk for one hour at room temperature The membrane was washed again with the same protocol as

P a ge I 3

above but with an extra step after the initial wash the membrane was placed in 1XTBS for 5 minutes Lastly before imaging the ECL (Electrochemiluminescence) reagent (Supersignal West stable peroxide solution mixed with Supersignal West pico luminolenhancer solution) was added to the membrane and rocked for 5 minutes After 5 minutes the membrane was imaged (Kodak Image Station 440)

TBS Tert-Butyldimethylsilyl Ether

Antibodies Used Santa Cruz Biotechnology MCT1 (T-19) Scshy14917 lot 01311 goat polyclonal IgG Santa Cruz Biotechnology IgG-HRP Sc-2020 Lot B0513 HRP conjugated Blocking Peptide Santa Crus Biotechnology MCTI (T-19)P Scshy14917 P Lot G 1212 Blocking Peptide

Data

10 9 8 7 5 4 3 2

--40

Figure 1 This is an image of the nitrocellulose membrane stained in Ponceau after the transfer While the lanes look more smeared than normal it is apparent that proteins residing at ~40 kDa are present

Figure 2 This image shows proteins from prepared muscle homogenates The left half shows non-centrifuged homogenates while the right half shows centrifuged homogenates (13000 rpm at room tern perature) Lanes 2 and 7 represent the protein samples prepared by using Santa Cruz reagents 5X loading buffer and then boiled for 3 minutes Lanes 3 and 8 represent the protocol for preparing homogenates portrayed in this paper Lanes 4 and 9 represent proteins of homogenates made using Santa Cruz Reagents Santa Cruz 2X buffer and boiled for 10 minutes Lanes 5 andlO represent proteins of homogenates made using the Santa Cruz reagents Santa Cruz 2X loading buffer BME and boiled for 10 minutes Lane 2 shows the most promise by presenting with three bands one at 40 kOa and two above 70 kOa It is possible the larger proteins exist as tetramers and dimers of the MCTI protein

Figure 3 This image shows a completed Western using different techniques for denaturation of the protein in order to increase

P a g e 14

separation The membrane on the right was treated with blocking peptides Lanes 2 and 7 represent proteins prepared using 2X SOSshyPAGE loading buffer and BME then boiled for 5 minutes Lanes 3 and 8 represent proteins prepared using 2X SOS-PAGE loading buffer and BME then heated to 37deg C for 30 minutes Lanes 4 and 9 represent proteins prepared using 2X SOS-PAGE loading buffer with BME and heated to 70deg C for 10 minutes Lanes 5 and 10 represent proteins prepared using 2X SOSshyPAGE loading buffer with OTT (Oithiothreitol) then boiled for 5 minutes This procedure did not result in convincing detection of the MCTl protein

Results

The Western pictured in figure 2 (lane 3) potentially shows the MCTI protein at ~40 kOa However two other bands also persist darker and more prominent than the band at 40kDa The other bands may in fact be dimers or tetramers of the MCTI protein which did not efficiently break apart during sample preparation In order to further degrade the MCT1 proteins a higher concentration of BME was experimented with along with different heating methods after the addition of BME One sample was even prepared with OTT instead of BME As figure 2 shows the higher concentrations of BME and the sample of OTT did not result in a single dark band at ~40 kOa To ensure the bands present were dimers or tetramers of the MCT1 protein a blocking peptide was added to a second membrane The bands seen in figure 3 did not disappear with the blocking peptide and therefore it is assumed the bands are not associated with the MCTI protein Another technique performed to isolate the MCTl protein from the interfering proteins included different centrifugation styles However as shown in figure 3 the centrifugation after homogenization completely eliminated any traces of the MCT1 protein

Discussion

Thus far troubleshooting with blocking peptides differing concentrations of BME the addition of OTT changes in heating temperatures and time the use of milk vs BSA

(Bovine Serum Albumin) and centrifugation techniques has not yielded viable results for determining the presence of MCTI in muscle Further troubleshooting includes the use of urea a positive control or a more sensitive ECL reagent Urea like SDS causes the further denaturation of proteins and a sample with overexpressed MCT 1 to act as a positive control to ensure the functionality of the antibody Possible changes in technique include longer exposure time for the membrane or the use of a tissue other than muscle As a last resort a new MCT 1 antibody should be tried Other research has proved the location and existence of the MCTI protein in muscle therefore the possibility stands that the antibody used in these experiments was faulty

Conclusions

Overall the troubleshooting performed during these experiments allowed for the betterment of the Homogenization preparation protocol and the Western protocol The troubleshooting also eliminated many procedures For example strong centrifugation after the addition of SDS buffer with BME results in the loss of the MCTI protein into the pellet Another a stronger concentration of BME or even switching to DTT does not break apart the tetramers and dimers of the MCT 1 protein Hopefully urea will do what SDS did not by breaking apart the tetramers and dimers to yield a protein band at ~40kDa and cross checked with the blocking peptide If the urea does not work the next step is to use a positive antibody as a positive control to confirm that the current antibody is attaching to the right protein Like mentioned previously a new antibody may have to be selected and the process of troubleshooting may have to start back at square one

Eventually the MCTI protein will be confirmed at ~40 kDa and mark the next step to uncovering the secret behind memory storage and recollection Further experiments will be performed comparing the level of the MCT1 proteins between differing mouse genotypes and the existing levels after differing behavioral activities The resulting comparisons and

P age I 5

analysis will allow insight into what occurs with the MCTI protein and lactate levels in the brain during hypoglycemia Medical fields dealing with diabetes Alzheimers disease or other metabolic deficiencies affecting cognitive function hope to benefit from this research and similar research in the near future

References

Amaral A 1 (2012) Effects of Hypoglycemia on Neuronal Metabolism in the Adult Brain Role of Alternative Substrates to Glucose Inherit Metab Dis Doi 101007510545-012-9553-3

Berg J M Tymoczko J L amp Stryer L (2012) BiochemistryNew York W H Freeman amp Company

Bezzi P amp Voltera A (2011) Astrocytes Pwering Memeroy Cell 144 Doi 101016jceI1201102027

Gold P E amp Korol D L (2012) Making Memories Matter Frontiers in Integrative Neuroscience 6(166) Doi 103389frint201200116

Newman L A Korol D L Gold P E (2011) Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing PLos ONE 6(12 Doi 10 13711joumalpone0028427

Suzuki A et al (2011) Astrocyte-Neuron Lactate Transport Is Required for Long Term Memory Formation Cell 144 810-823 Doi 101016jceI1201102018

Completion Report httpsllwwwcitiprogramorgmemberslearnersllicrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with Mice in Research Settings Curriculum Completion Report Printed on 5152013

Leamer Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Working with Mice in Research

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343726)

Elective Modules Date

Completed Score Introduction to Working with Mice in Research Settings 051513 no quiz

Research Mandates and Occupational Health Issues 051513 313 (1 00 )

Alternatives Searches Humane Standards Housing and Acclimation and Quarantine

0511513 212 (100)

Detecting Pain and Distress Genetics and Biological Features

0511513 313 (100)

Injections Blood Collection and Antibody Production 0511513 212 (10000)

Surgery Supportive Care and Monitoring Euthanasia and References

051513 415 (80)

For this Completion Report to be valid the learner Ilisted above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

Return

1 of 1 51512013 5 18 PM

memory formation Further proving the importance of lactate blockage of monocarboxy late transporters (lactate transporters) causes amnesia in test subjects (Suzuki 2011) The increase in extracellular lactate occurs through the glycogenolysis of intercellular glycogen or through the glycolysis of extracellular glucose which is seen to decrease during spatial learning (Newman and Gold 2011) The process of glycogenolysis occurs in the astrocyte where lactate is produced It is then presumed that lactate is transported into neurons to support further metabolic functions (Newman and Gold) The MCTI and MCT2 proteins focused on in this project represent the order of transportation MCTI is an astrocytic transporter and MCT2 acts as a neuronal transporter of lactate The measurement of lactate transport in and out of the astrocytesneurons can be related to the relative amounts of MCTI and MCT2 proteins When a specific metabolic process is in high demand so are the proteins (Bezzi 2011)

After the MCTI and MCT2 proteins are reliably detected the relative amounts will be compared in three different genotypes of mice as well as the levels (through observation of Western data) after varying physical and mental tests The types of mice used include HO HOG and wild type mice Wild type mice exhibit the nonnal expression of glycogen synthase so nonnal levels of glycogen are present in muscle and liver tissue HO mice on the other hand are genetically engineered to lack glycogen synthase so no glycogen stores can be found in their muscle or brain HOG mice are also engineered to lack glycogen synthase in brain tissue but overproduce glycogen in muscle A few of the tests perfonned on the mice include wire hang rotarod object recognition and passive avoidance The wire hang measures the endurance of the mouse in accordance to the amount of available glycogen in its muscles The rotarod measures motor learning The object recognition test focuses on recognition and the passive avoidance tests measures associative memory

Discovering the role of glucose glycogen and lactate in the metabolic process of memory

Page 12

storage and recollection may influence the treatment of patients suffering from hypoglycemia Alzheimers disease or other diseases which effect memory Thus far some studies have determined that injections of epinephrine glucose and lactate increase memory storage and recollection in mice (Gold and Korol 2012)

Methods

Homo enization

The first step in detennining the relative amounts of lactate transporters prevalent in mouse brain tissue after spatial learning exercises and hypoglycemiahomeostasis is to establish a reliable method for detection This particular set of methods and data focus on optimizing the protocol for preparing tissue homogenates and perfonning Western blots in accordance with detecting MCTI Besides its location in astrocytes MCTI is also located in larger quantities throughout the body so for the purpose of detection mouse muscle tissue was homogenized and used

Tissue homogenization and preparation was tried several different ways The following procedure was proven to yield the best results but still requires modification to ensure the proteins presence First mice were sacrificed using cervical dislocation to reduce the amount of metabolites lost and then leg muscles were harvested The muscles were immediately placed in nitrogen for preservation Further preservation required storage in a -80degC freezer During tissue preparation for homogenization samples were also kept in nitrogen The tissue which was to be homogenized and prepared for future Bradford Assays and Western Blots was weighed out and homogenized using a buffer made up of

1 ml-SOmM Tris pH 7810 mM EDTA2mM EGTA1 OOmM NaF

37111 10 mgmL TLCK 2 ilL 1 M benzamidine SilL 100 mM PMSF 35 ilL BME 1 ilL 10 mgm L leupeptin

The samples were then centrifuged for 5 min at 3600xg at 4degC The supernatant was saved for Bradfords and Westerns Supernatant meant for Westerns was prepared with 5X SDS-PAGE loading buffer containing BME (2shymercaptoethanol) The samples were then boiled for 5 minutes then placed in -20deg C storage

EDTA Ethylenediaminetetraacetic acid EGTA Ethy lene-bis( oxyethy lenenitri 10)tetraacetic acid TLCK Tosyllysine chloromethylketone PMSF Phenylmethylsulfonyl Fluoride

SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)

The Western protocol focused on detecting MCT1 at 40-48 kDa Once again several protocols were used to detect the protein but the following protocol produced the best results Like mentioned before further modifications are necessary to affirm the presence of the MCT1 protein Using Bio-Rad Mini-PROTEAN TGX (cat456-1033) 1000 precast gels wells were loaded with 20lL of protein and the appropriate amounts of 5X buffer and homogenization buffer The gel was run for ~35 min at a voltage of 200 Next the proteins from the gel were transferred onto a nitrocellulose membrane See figure 1 as an example of how the transfer worked

Western

The membrane was then blocked overnight at 4deg C in lXTBSTweenl400 milk Next the membrane was exposed to a 1 200 MCTI dilution in 1XTBS005tween-20500milk for an hour at room temperature To determine the proteins bound by the antibodies blocking peptides were used The membrane was then washed with 1 XTBSTween two times for a few seconds each then three times for ten minutes each The membrane was then exposed to a 1 1 0000 dilution of the secondary anti-goat antibody in lXTBS00500Tween500milk for one hour at room temperature The membrane was washed again with the same protocol as

P a ge I 3

above but with an extra step after the initial wash the membrane was placed in 1XTBS for 5 minutes Lastly before imaging the ECL (Electrochemiluminescence) reagent (Supersignal West stable peroxide solution mixed with Supersignal West pico luminolenhancer solution) was added to the membrane and rocked for 5 minutes After 5 minutes the membrane was imaged (Kodak Image Station 440)

TBS Tert-Butyldimethylsilyl Ether

Antibodies Used Santa Cruz Biotechnology MCT1 (T-19) Scshy14917 lot 01311 goat polyclonal IgG Santa Cruz Biotechnology IgG-HRP Sc-2020 Lot B0513 HRP conjugated Blocking Peptide Santa Crus Biotechnology MCTI (T-19)P Scshy14917 P Lot G 1212 Blocking Peptide

Data

10 9 8 7 5 4 3 2

--40

Figure 1 This is an image of the nitrocellulose membrane stained in Ponceau after the transfer While the lanes look more smeared than normal it is apparent that proteins residing at ~40 kDa are present

Figure 2 This image shows proteins from prepared muscle homogenates The left half shows non-centrifuged homogenates while the right half shows centrifuged homogenates (13000 rpm at room tern perature) Lanes 2 and 7 represent the protein samples prepared by using Santa Cruz reagents 5X loading buffer and then boiled for 3 minutes Lanes 3 and 8 represent the protocol for preparing homogenates portrayed in this paper Lanes 4 and 9 represent proteins of homogenates made using Santa Cruz Reagents Santa Cruz 2X buffer and boiled for 10 minutes Lanes 5 andlO represent proteins of homogenates made using the Santa Cruz reagents Santa Cruz 2X loading buffer BME and boiled for 10 minutes Lane 2 shows the most promise by presenting with three bands one at 40 kOa and two above 70 kOa It is possible the larger proteins exist as tetramers and dimers of the MCTI protein

Figure 3 This image shows a completed Western using different techniques for denaturation of the protein in order to increase

P a g e 14

separation The membrane on the right was treated with blocking peptides Lanes 2 and 7 represent proteins prepared using 2X SOSshyPAGE loading buffer and BME then boiled for 5 minutes Lanes 3 and 8 represent proteins prepared using 2X SOS-PAGE loading buffer and BME then heated to 37deg C for 30 minutes Lanes 4 and 9 represent proteins prepared using 2X SOS-PAGE loading buffer with BME and heated to 70deg C for 10 minutes Lanes 5 and 10 represent proteins prepared using 2X SOSshyPAGE loading buffer with OTT (Oithiothreitol) then boiled for 5 minutes This procedure did not result in convincing detection of the MCTl protein

Results

The Western pictured in figure 2 (lane 3) potentially shows the MCTI protein at ~40 kOa However two other bands also persist darker and more prominent than the band at 40kDa The other bands may in fact be dimers or tetramers of the MCTI protein which did not efficiently break apart during sample preparation In order to further degrade the MCT1 proteins a higher concentration of BME was experimented with along with different heating methods after the addition of BME One sample was even prepared with OTT instead of BME As figure 2 shows the higher concentrations of BME and the sample of OTT did not result in a single dark band at ~40 kOa To ensure the bands present were dimers or tetramers of the MCT1 protein a blocking peptide was added to a second membrane The bands seen in figure 3 did not disappear with the blocking peptide and therefore it is assumed the bands are not associated with the MCTI protein Another technique performed to isolate the MCTl protein from the interfering proteins included different centrifugation styles However as shown in figure 3 the centrifugation after homogenization completely eliminated any traces of the MCT1 protein

Discussion

Thus far troubleshooting with blocking peptides differing concentrations of BME the addition of OTT changes in heating temperatures and time the use of milk vs BSA

(Bovine Serum Albumin) and centrifugation techniques has not yielded viable results for determining the presence of MCTI in muscle Further troubleshooting includes the use of urea a positive control or a more sensitive ECL reagent Urea like SDS causes the further denaturation of proteins and a sample with overexpressed MCT 1 to act as a positive control to ensure the functionality of the antibody Possible changes in technique include longer exposure time for the membrane or the use of a tissue other than muscle As a last resort a new MCT 1 antibody should be tried Other research has proved the location and existence of the MCTI protein in muscle therefore the possibility stands that the antibody used in these experiments was faulty

Conclusions

Overall the troubleshooting performed during these experiments allowed for the betterment of the Homogenization preparation protocol and the Western protocol The troubleshooting also eliminated many procedures For example strong centrifugation after the addition of SDS buffer with BME results in the loss of the MCTI protein into the pellet Another a stronger concentration of BME or even switching to DTT does not break apart the tetramers and dimers of the MCT 1 protein Hopefully urea will do what SDS did not by breaking apart the tetramers and dimers to yield a protein band at ~40kDa and cross checked with the blocking peptide If the urea does not work the next step is to use a positive antibody as a positive control to confirm that the current antibody is attaching to the right protein Like mentioned previously a new antibody may have to be selected and the process of troubleshooting may have to start back at square one

Eventually the MCTI protein will be confirmed at ~40 kDa and mark the next step to uncovering the secret behind memory storage and recollection Further experiments will be performed comparing the level of the MCT1 proteins between differing mouse genotypes and the existing levels after differing behavioral activities The resulting comparisons and

P age I 5

analysis will allow insight into what occurs with the MCTI protein and lactate levels in the brain during hypoglycemia Medical fields dealing with diabetes Alzheimers disease or other metabolic deficiencies affecting cognitive function hope to benefit from this research and similar research in the near future

References

Amaral A 1 (2012) Effects of Hypoglycemia on Neuronal Metabolism in the Adult Brain Role of Alternative Substrates to Glucose Inherit Metab Dis Doi 101007510545-012-9553-3

Berg J M Tymoczko J L amp Stryer L (2012) BiochemistryNew York W H Freeman amp Company

Bezzi P amp Voltera A (2011) Astrocytes Pwering Memeroy Cell 144 Doi 101016jceI1201102027

Gold P E amp Korol D L (2012) Making Memories Matter Frontiers in Integrative Neuroscience 6(166) Doi 103389frint201200116

Newman L A Korol D L Gold P E (2011) Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing PLos ONE 6(12 Doi 10 13711joumalpone0028427

Suzuki A et al (2011) Astrocyte-Neuron Lactate Transport Is Required for Long Term Memory Formation Cell 144 810-823 Doi 101016jceI1201102018

Completion Report httpsllwwwcitiprogramorgmemberslearnersllicrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with Mice in Research Settings Curriculum Completion Report Printed on 5152013

Leamer Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Working with Mice in Research

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343726)

Elective Modules Date

Completed Score Introduction to Working with Mice in Research Settings 051513 no quiz

Research Mandates and Occupational Health Issues 051513 313 (1 00 )

Alternatives Searches Humane Standards Housing and Acclimation and Quarantine

0511513 212 (100)

Detecting Pain and Distress Genetics and Biological Features

0511513 313 (100)

Injections Blood Collection and Antibody Production 0511513 212 (10000)

Surgery Supportive Care and Monitoring Euthanasia and References

051513 415 (80)

For this Completion Report to be valid the learner Ilisted above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

Return

1 of 1 51512013 5 18 PM

The samples were then centrifuged for 5 min at 3600xg at 4degC The supernatant was saved for Bradfords and Westerns Supernatant meant for Westerns was prepared with 5X SDS-PAGE loading buffer containing BME (2shymercaptoethanol) The samples were then boiled for 5 minutes then placed in -20deg C storage

EDTA Ethylenediaminetetraacetic acid EGTA Ethy lene-bis( oxyethy lenenitri 10)tetraacetic acid TLCK Tosyllysine chloromethylketone PMSF Phenylmethylsulfonyl Fluoride

SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)

The Western protocol focused on detecting MCT1 at 40-48 kDa Once again several protocols were used to detect the protein but the following protocol produced the best results Like mentioned before further modifications are necessary to affirm the presence of the MCT1 protein Using Bio-Rad Mini-PROTEAN TGX (cat456-1033) 1000 precast gels wells were loaded with 20lL of protein and the appropriate amounts of 5X buffer and homogenization buffer The gel was run for ~35 min at a voltage of 200 Next the proteins from the gel were transferred onto a nitrocellulose membrane See figure 1 as an example of how the transfer worked

Western

The membrane was then blocked overnight at 4deg C in lXTBSTweenl400 milk Next the membrane was exposed to a 1 200 MCTI dilution in 1XTBS005tween-20500milk for an hour at room temperature To determine the proteins bound by the antibodies blocking peptides were used The membrane was then washed with 1 XTBSTween two times for a few seconds each then three times for ten minutes each The membrane was then exposed to a 1 1 0000 dilution of the secondary anti-goat antibody in lXTBS00500Tween500milk for one hour at room temperature The membrane was washed again with the same protocol as

P a ge I 3

above but with an extra step after the initial wash the membrane was placed in 1XTBS for 5 minutes Lastly before imaging the ECL (Electrochemiluminescence) reagent (Supersignal West stable peroxide solution mixed with Supersignal West pico luminolenhancer solution) was added to the membrane and rocked for 5 minutes After 5 minutes the membrane was imaged (Kodak Image Station 440)

TBS Tert-Butyldimethylsilyl Ether

Antibodies Used Santa Cruz Biotechnology MCT1 (T-19) Scshy14917 lot 01311 goat polyclonal IgG Santa Cruz Biotechnology IgG-HRP Sc-2020 Lot B0513 HRP conjugated Blocking Peptide Santa Crus Biotechnology MCTI (T-19)P Scshy14917 P Lot G 1212 Blocking Peptide

Data

10 9 8 7 5 4 3 2

--40

Figure 1 This is an image of the nitrocellulose membrane stained in Ponceau after the transfer While the lanes look more smeared than normal it is apparent that proteins residing at ~40 kDa are present

Figure 2 This image shows proteins from prepared muscle homogenates The left half shows non-centrifuged homogenates while the right half shows centrifuged homogenates (13000 rpm at room tern perature) Lanes 2 and 7 represent the protein samples prepared by using Santa Cruz reagents 5X loading buffer and then boiled for 3 minutes Lanes 3 and 8 represent the protocol for preparing homogenates portrayed in this paper Lanes 4 and 9 represent proteins of homogenates made using Santa Cruz Reagents Santa Cruz 2X buffer and boiled for 10 minutes Lanes 5 andlO represent proteins of homogenates made using the Santa Cruz reagents Santa Cruz 2X loading buffer BME and boiled for 10 minutes Lane 2 shows the most promise by presenting with three bands one at 40 kOa and two above 70 kOa It is possible the larger proteins exist as tetramers and dimers of the MCTI protein

Figure 3 This image shows a completed Western using different techniques for denaturation of the protein in order to increase

P a g e 14

separation The membrane on the right was treated with blocking peptides Lanes 2 and 7 represent proteins prepared using 2X SOSshyPAGE loading buffer and BME then boiled for 5 minutes Lanes 3 and 8 represent proteins prepared using 2X SOS-PAGE loading buffer and BME then heated to 37deg C for 30 minutes Lanes 4 and 9 represent proteins prepared using 2X SOS-PAGE loading buffer with BME and heated to 70deg C for 10 minutes Lanes 5 and 10 represent proteins prepared using 2X SOSshyPAGE loading buffer with OTT (Oithiothreitol) then boiled for 5 minutes This procedure did not result in convincing detection of the MCTl protein

Results

The Western pictured in figure 2 (lane 3) potentially shows the MCTI protein at ~40 kOa However two other bands also persist darker and more prominent than the band at 40kDa The other bands may in fact be dimers or tetramers of the MCTI protein which did not efficiently break apart during sample preparation In order to further degrade the MCT1 proteins a higher concentration of BME was experimented with along with different heating methods after the addition of BME One sample was even prepared with OTT instead of BME As figure 2 shows the higher concentrations of BME and the sample of OTT did not result in a single dark band at ~40 kOa To ensure the bands present were dimers or tetramers of the MCT1 protein a blocking peptide was added to a second membrane The bands seen in figure 3 did not disappear with the blocking peptide and therefore it is assumed the bands are not associated with the MCTI protein Another technique performed to isolate the MCTl protein from the interfering proteins included different centrifugation styles However as shown in figure 3 the centrifugation after homogenization completely eliminated any traces of the MCT1 protein

Discussion

Thus far troubleshooting with blocking peptides differing concentrations of BME the addition of OTT changes in heating temperatures and time the use of milk vs BSA

(Bovine Serum Albumin) and centrifugation techniques has not yielded viable results for determining the presence of MCTI in muscle Further troubleshooting includes the use of urea a positive control or a more sensitive ECL reagent Urea like SDS causes the further denaturation of proteins and a sample with overexpressed MCT 1 to act as a positive control to ensure the functionality of the antibody Possible changes in technique include longer exposure time for the membrane or the use of a tissue other than muscle As a last resort a new MCT 1 antibody should be tried Other research has proved the location and existence of the MCTI protein in muscle therefore the possibility stands that the antibody used in these experiments was faulty

Conclusions

Overall the troubleshooting performed during these experiments allowed for the betterment of the Homogenization preparation protocol and the Western protocol The troubleshooting also eliminated many procedures For example strong centrifugation after the addition of SDS buffer with BME results in the loss of the MCTI protein into the pellet Another a stronger concentration of BME or even switching to DTT does not break apart the tetramers and dimers of the MCT 1 protein Hopefully urea will do what SDS did not by breaking apart the tetramers and dimers to yield a protein band at ~40kDa and cross checked with the blocking peptide If the urea does not work the next step is to use a positive antibody as a positive control to confirm that the current antibody is attaching to the right protein Like mentioned previously a new antibody may have to be selected and the process of troubleshooting may have to start back at square one

Eventually the MCTI protein will be confirmed at ~40 kDa and mark the next step to uncovering the secret behind memory storage and recollection Further experiments will be performed comparing the level of the MCT1 proteins between differing mouse genotypes and the existing levels after differing behavioral activities The resulting comparisons and

P age I 5

analysis will allow insight into what occurs with the MCTI protein and lactate levels in the brain during hypoglycemia Medical fields dealing with diabetes Alzheimers disease or other metabolic deficiencies affecting cognitive function hope to benefit from this research and similar research in the near future

References

Amaral A 1 (2012) Effects of Hypoglycemia on Neuronal Metabolism in the Adult Brain Role of Alternative Substrates to Glucose Inherit Metab Dis Doi 101007510545-012-9553-3

Berg J M Tymoczko J L amp Stryer L (2012) BiochemistryNew York W H Freeman amp Company

Bezzi P amp Voltera A (2011) Astrocytes Pwering Memeroy Cell 144 Doi 101016jceI1201102027

Gold P E amp Korol D L (2012) Making Memories Matter Frontiers in Integrative Neuroscience 6(166) Doi 103389frint201200116

Newman L A Korol D L Gold P E (2011) Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing PLos ONE 6(12 Doi 10 13711joumalpone0028427

Suzuki A et al (2011) Astrocyte-Neuron Lactate Transport Is Required for Long Term Memory Formation Cell 144 810-823 Doi 101016jceI1201102018

Completion Report httpsllwwwcitiprogramorgmemberslearnersllicrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with Mice in Research Settings Curriculum Completion Report Printed on 5152013

Leamer Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Working with Mice in Research

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343726)

Elective Modules Date

Completed Score Introduction to Working with Mice in Research Settings 051513 no quiz

Research Mandates and Occupational Health Issues 051513 313 (1 00 )

Alternatives Searches Humane Standards Housing and Acclimation and Quarantine

0511513 212 (100)

Detecting Pain and Distress Genetics and Biological Features

0511513 313 (100)

Injections Blood Collection and Antibody Production 0511513 212 (10000)

Surgery Supportive Care and Monitoring Euthanasia and References

051513 415 (80)

For this Completion Report to be valid the learner Ilisted above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

Return

1 of 1 51512013 5 18 PM

Figure 2 This image shows proteins from prepared muscle homogenates The left half shows non-centrifuged homogenates while the right half shows centrifuged homogenates (13000 rpm at room tern perature) Lanes 2 and 7 represent the protein samples prepared by using Santa Cruz reagents 5X loading buffer and then boiled for 3 minutes Lanes 3 and 8 represent the protocol for preparing homogenates portrayed in this paper Lanes 4 and 9 represent proteins of homogenates made using Santa Cruz Reagents Santa Cruz 2X buffer and boiled for 10 minutes Lanes 5 andlO represent proteins of homogenates made using the Santa Cruz reagents Santa Cruz 2X loading buffer BME and boiled for 10 minutes Lane 2 shows the most promise by presenting with three bands one at 40 kOa and two above 70 kOa It is possible the larger proteins exist as tetramers and dimers of the MCTI protein

Figure 3 This image shows a completed Western using different techniques for denaturation of the protein in order to increase

P a g e 14

separation The membrane on the right was treated with blocking peptides Lanes 2 and 7 represent proteins prepared using 2X SOSshyPAGE loading buffer and BME then boiled for 5 minutes Lanes 3 and 8 represent proteins prepared using 2X SOS-PAGE loading buffer and BME then heated to 37deg C for 30 minutes Lanes 4 and 9 represent proteins prepared using 2X SOS-PAGE loading buffer with BME and heated to 70deg C for 10 minutes Lanes 5 and 10 represent proteins prepared using 2X SOSshyPAGE loading buffer with OTT (Oithiothreitol) then boiled for 5 minutes This procedure did not result in convincing detection of the MCTl protein

Results

The Western pictured in figure 2 (lane 3) potentially shows the MCTI protein at ~40 kOa However two other bands also persist darker and more prominent than the band at 40kDa The other bands may in fact be dimers or tetramers of the MCTI protein which did not efficiently break apart during sample preparation In order to further degrade the MCT1 proteins a higher concentration of BME was experimented with along with different heating methods after the addition of BME One sample was even prepared with OTT instead of BME As figure 2 shows the higher concentrations of BME and the sample of OTT did not result in a single dark band at ~40 kOa To ensure the bands present were dimers or tetramers of the MCT1 protein a blocking peptide was added to a second membrane The bands seen in figure 3 did not disappear with the blocking peptide and therefore it is assumed the bands are not associated with the MCTI protein Another technique performed to isolate the MCTl protein from the interfering proteins included different centrifugation styles However as shown in figure 3 the centrifugation after homogenization completely eliminated any traces of the MCT1 protein

Discussion

Thus far troubleshooting with blocking peptides differing concentrations of BME the addition of OTT changes in heating temperatures and time the use of milk vs BSA

(Bovine Serum Albumin) and centrifugation techniques has not yielded viable results for determining the presence of MCTI in muscle Further troubleshooting includes the use of urea a positive control or a more sensitive ECL reagent Urea like SDS causes the further denaturation of proteins and a sample with overexpressed MCT 1 to act as a positive control to ensure the functionality of the antibody Possible changes in technique include longer exposure time for the membrane or the use of a tissue other than muscle As a last resort a new MCT 1 antibody should be tried Other research has proved the location and existence of the MCTI protein in muscle therefore the possibility stands that the antibody used in these experiments was faulty

Conclusions

Overall the troubleshooting performed during these experiments allowed for the betterment of the Homogenization preparation protocol and the Western protocol The troubleshooting also eliminated many procedures For example strong centrifugation after the addition of SDS buffer with BME results in the loss of the MCTI protein into the pellet Another a stronger concentration of BME or even switching to DTT does not break apart the tetramers and dimers of the MCT 1 protein Hopefully urea will do what SDS did not by breaking apart the tetramers and dimers to yield a protein band at ~40kDa and cross checked with the blocking peptide If the urea does not work the next step is to use a positive antibody as a positive control to confirm that the current antibody is attaching to the right protein Like mentioned previously a new antibody may have to be selected and the process of troubleshooting may have to start back at square one

Eventually the MCTI protein will be confirmed at ~40 kDa and mark the next step to uncovering the secret behind memory storage and recollection Further experiments will be performed comparing the level of the MCT1 proteins between differing mouse genotypes and the existing levels after differing behavioral activities The resulting comparisons and

P age I 5

analysis will allow insight into what occurs with the MCTI protein and lactate levels in the brain during hypoglycemia Medical fields dealing with diabetes Alzheimers disease or other metabolic deficiencies affecting cognitive function hope to benefit from this research and similar research in the near future

References

Amaral A 1 (2012) Effects of Hypoglycemia on Neuronal Metabolism in the Adult Brain Role of Alternative Substrates to Glucose Inherit Metab Dis Doi 101007510545-012-9553-3

Berg J M Tymoczko J L amp Stryer L (2012) BiochemistryNew York W H Freeman amp Company

Bezzi P amp Voltera A (2011) Astrocytes Pwering Memeroy Cell 144 Doi 101016jceI1201102027

Gold P E amp Korol D L (2012) Making Memories Matter Frontiers in Integrative Neuroscience 6(166) Doi 103389frint201200116

Newman L A Korol D L Gold P E (2011) Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing PLos ONE 6(12 Doi 10 13711joumalpone0028427

Suzuki A et al (2011) Astrocyte-Neuron Lactate Transport Is Required for Long Term Memory Formation Cell 144 810-823 Doi 101016jceI1201102018

Completion Report httpsllwwwcitiprogramorgmemberslearnersllicrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with Mice in Research Settings Curriculum Completion Report Printed on 5152013

Leamer Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Working with Mice in Research

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343726)

Elective Modules Date

Completed Score Introduction to Working with Mice in Research Settings 051513 no quiz

Research Mandates and Occupational Health Issues 051513 313 (1 00 )

Alternatives Searches Humane Standards Housing and Acclimation and Quarantine

0511513 212 (100)

Detecting Pain and Distress Genetics and Biological Features

0511513 313 (100)

Injections Blood Collection and Antibody Production 0511513 212 (10000)

Surgery Supportive Care and Monitoring Euthanasia and References

051513 415 (80)

For this Completion Report to be valid the learner Ilisted above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

Return

1 of 1 51512013 5 18 PM

(Bovine Serum Albumin) and centrifugation techniques has not yielded viable results for determining the presence of MCTI in muscle Further troubleshooting includes the use of urea a positive control or a more sensitive ECL reagent Urea like SDS causes the further denaturation of proteins and a sample with overexpressed MCT 1 to act as a positive control to ensure the functionality of the antibody Possible changes in technique include longer exposure time for the membrane or the use of a tissue other than muscle As a last resort a new MCT 1 antibody should be tried Other research has proved the location and existence of the MCTI protein in muscle therefore the possibility stands that the antibody used in these experiments was faulty

Conclusions

Overall the troubleshooting performed during these experiments allowed for the betterment of the Homogenization preparation protocol and the Western protocol The troubleshooting also eliminated many procedures For example strong centrifugation after the addition of SDS buffer with BME results in the loss of the MCTI protein into the pellet Another a stronger concentration of BME or even switching to DTT does not break apart the tetramers and dimers of the MCT 1 protein Hopefully urea will do what SDS did not by breaking apart the tetramers and dimers to yield a protein band at ~40kDa and cross checked with the blocking peptide If the urea does not work the next step is to use a positive antibody as a positive control to confirm that the current antibody is attaching to the right protein Like mentioned previously a new antibody may have to be selected and the process of troubleshooting may have to start back at square one

Eventually the MCTI protein will be confirmed at ~40 kDa and mark the next step to uncovering the secret behind memory storage and recollection Further experiments will be performed comparing the level of the MCT1 proteins between differing mouse genotypes and the existing levels after differing behavioral activities The resulting comparisons and

P age I 5

analysis will allow insight into what occurs with the MCTI protein and lactate levels in the brain during hypoglycemia Medical fields dealing with diabetes Alzheimers disease or other metabolic deficiencies affecting cognitive function hope to benefit from this research and similar research in the near future

References

Amaral A 1 (2012) Effects of Hypoglycemia on Neuronal Metabolism in the Adult Brain Role of Alternative Substrates to Glucose Inherit Metab Dis Doi 101007510545-012-9553-3

Berg J M Tymoczko J L amp Stryer L (2012) BiochemistryNew York W H Freeman amp Company

Bezzi P amp Voltera A (2011) Astrocytes Pwering Memeroy Cell 144 Doi 101016jceI1201102027

Gold P E amp Korol D L (2012) Making Memories Matter Frontiers in Integrative Neuroscience 6(166) Doi 103389frint201200116

Newman L A Korol D L Gold P E (2011) Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing PLos ONE 6(12 Doi 10 13711joumalpone0028427

Suzuki A et al (2011) Astrocyte-Neuron Lactate Transport Is Required for Long Term Memory Formation Cell 144 810-823 Doi 101016jceI1201102018

Completion Report httpsllwwwcitiprogramorgmemberslearnersllicrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with Mice in Research Settings Curriculum Completion Report Printed on 5152013

Leamer Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Working with Mice in Research

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343726)

Elective Modules Date

Completed Score Introduction to Working with Mice in Research Settings 051513 no quiz

Research Mandates and Occupational Health Issues 051513 313 (1 00 )

Alternatives Searches Humane Standards Housing and Acclimation and Quarantine

0511513 212 (100)

Detecting Pain and Distress Genetics and Biological Features

0511513 313 (100)

Injections Blood Collection and Antibody Production 0511513 212 (10000)

Surgery Supportive Care and Monitoring Euthanasia and References

051513 415 (80)

For this Completion Report to be valid the learner Ilisted above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

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1 of 1 51512013 5 18 PM

Completion Report httpsllwwwcitiprogramorgmemberslearnersllicrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with Mice in Research Settings Curriculum Completion Report Printed on 5152013

Leamer Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Working with Mice in Research

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343726)

Elective Modules Date

Completed Score Introduction to Working with Mice in Research Settings 051513 no quiz

Research Mandates and Occupational Health Issues 051513 313 (1 00 )

Alternatives Searches Humane Standards Housing and Acclimation and Quarantine

0511513 212 (100)

Detecting Pain and Distress Genetics and Biological Features

0511513 313 (100)

Injections Blood Collection and Antibody Production 0511513 212 (10000)

Surgery Supportive Care and Monitoring Euthanasia and References

051513 415 (80)

For this Completion Report to be valid the learner Ilisted above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

Return

1 of 1 51512013 5 18 PM

Completion Report httpsllwwwcitiprogramorgimemberslearnersWcrbystageaspstrKe

CITI Collaborative Institutional Training Initiative (CITI)

Working with the IACUC Curriculum Completion Report Printed on 5152013

Learner Audrey Aldrich (username aealdrich92) Institution Ball State University Contact Information 3651 N Marleon Drive

Apt 201 Muncie Indiana 47304 United States Department Biochemistry Phone 26-920-4782 Email aealdrichbsuedu

Investigators Staff and Students The CITI Basic Course in Laboratory Animal Welfare for Investigators Staff and Students

Stage 1 Lab Animal Research Passed on 051513 (Ref 10343725)

Elective Modules Date

Completed Score

Introduction to Working with the IACUC 051513 no quiz

Working with the IACUC 051513 313 (100 )

Federal Mandates 0511513 414 (1 0000)

The Veterinary Consultation 051513 111 (100)

Getting Started 0511513 515 (100)

~Iternatives 0511513 818 (10000)

~voiding Unnecessary Duplication 051513 111 (100)

USDA PainlDistress Categories 051513 818 (1 0000)

Endpoint Criteria 051513 212 (100)

Surgery 051513 1111 (100) Antibody Production 051513 no quiz

Collecting Blood Samples 051513 313 (1 0000)

Personnel Training and Experience 051513 313 (1 0000)

Occupational Health and Safety 051513 111 (100) Using Hazardous and Toxic Agents in Animals 051513 616 (10000)

Housing Social Animals 051513 111 (100)

Housing Rodents on Wire Floors 051513 111 (10000)

Dog Exercise 051513 111 (10000) Primate Psychological Enrichment 051513 111 (10000) Prolonged Restraint 051513 212 (10000) Euthanasia 051513 818 (100) Using Human Patient Care Areas for Animal Research 051513 111 (100

)

Using Explosive Agents in the Animal Facility 0511513 111 (10000)

1 of _ 1 ~011 S~01 PM

Completion Report httpswwwcitiprogramorgmemberslearnersIIJcrbystageaspstrKe

Making Changes after You Receive Approval 051513 111 (100)

Reporting Misuse Mistreatment or Non-Compliance 051513 no quiz

Final Comments 051513 no quiz

For this Completion Report to be valid the learner listed above must be affiliated with a CITI participating institution Falsified information and unauthorized use of the CITI course site is unethical and may be considered scientific misconduct by your institution

Paul Braunschweiger PhD Professor University of Miami Director Office of Research Education CITI Course Coordinator

Return

2of2 51152013 503 PM