Changing stroke rehab and research worldwide now.Time is Brain! trillions and trillions of neurons that DIE each day because there are NO effective hyperacute therapies besides tPA(only 12% effective). I have 523 posts on hyperacute therapy, enough for researchers to spend decades proving them out. These are my personal ideas and blog on stroke rehabilitation and stroke research. Do not attempt any of these without checking with your medical provider. Unless you join me in agitating, when you need these therapies they won't be there.

What this blog is for:

My blog is not to help survivors recover, it is to have the 10 million yearly stroke survivors light fires underneath their doctors, stroke hospitals and stroke researchers to get stroke solved. 100% recovery. The stroke medical world is completely failing at that goal, they don't even have it as a goal. Shortly after getting out of the hospital and getting NO information on the process or protocols of stroke rehabilitation and recovery I started searching on the internet and found that no other survivor received useful information. This is an attempt to cover all stroke rehabilitation information that should be readily available to survivors so they can talk with informed knowledge to their medical staff. It lays out what needs to be done to get stroke survivors closer to 100% recovery. It's quite disgusting that this information is not available from every stroke association and doctors group.

Wednesday, July 26, 2023

Muscle-building supplement β-hydroxy β-methylbutyrate binds to PPARα to improve hippocampal functions in mice

Something in this research says this: A muscle-building supplement known as beta-hydroxy beta-methylbutyrate (HMB) improved spatial learning and memory in a mouse model of Alzheimer's disease. (Cell Reports).

I won't be doing this until it is safely proven in humans.

 

Muscle-building supplement β-hydroxy β-methylbutyrate binds to PPARα to improve hippocampal functions in mice

Open AccessPublished:July 11, 2023DOI:https://doi.org/10.1016/j.celrep.2023.112717

Highlights

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    Muscle-building supplement HMB binds to PPARα
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    HMB increases morphological plasticity of hippocampal neurons via PPARα
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    Oral HMB improves hippocampal functions in 5XFAD mice using PPARα
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    Oral HMB lowers plaques in 5XFAD mice through PPARα

Summary

This study underlines the importance of β-hydroxy β-methylbutyrate (HMB), a muscle-building supplement in human, in increasing mouse hippocampal plasticity. Detailed proteomic analyses reveal that HMB serves as a ligand of peroxisome proliferator-activated receptor α (PPARα), a nuclear hormone receptor involved in fat metabolism, via interaction with the Y314 residue. Accordingly, HMB is ineffective in increasing plasticity of PPARα−/− hippocampal neurons. While lentiviral establishment of full-length PPARα restores the plasticity-promoting effect of HMB in PPARα−/− hippocampal neurons, lentiviral transduction of Y314D-PPARα remains unable to do that, highlighting the importance of HMB’s interaction with the Y314 residue. Additionally, oral HMB improves spatial learning and memory and reduces plaque load in 5X familial Alzheimer’s disease (5XFAD) mice, but not in 5XFADΔPPARα mice (5XFAD lacking PPARα), indicating the involvement of PPARα in HMB-mediated neuroprotection in 5XFAD mice. These results delineate neuroprotective functions of HMB and suggest that this widely used supplement may be repurposed for AD.

Graphical abstract

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Research topic(s)

Introduction

Alzheimer’s disease (AD) is the most common human neurodegenerative disorder, comprising almost two-thirds of all cases of dementia. For patients with AD, usually the first clinical sign appears after age 60. Although the etiology of AD is poorly understood, it is now well established that AD is a multifactorial disease of the brain involving lifestyle, genetic, and environmental factors., Senile plaques, neurofibrillary tangles, and neuronal loss are classical pathological features of AD. However, synapse loss is believed to be a profound neuropathology of AD, and accordingly, from a clinical angle, it is identified by progressive impairment in memory, judgment, decision-making, and language usage. It has been reported that individuals with early AD have significantly fewer synapses than those with mild cognitive impairment (MCI) and no cognitive impairment (NCI) and that the number of synapses exhibits a significant correlation with the subject’s Mini-Mental State scores. Interestingly, synaptic loss does not display any relationship to either Braak stage or apoE genotype. Therefore, promotion of hippocampal plasticity is an important area of research, as it may help in the preservation of memory in healthy brains and improvement in cognitive functions in individuals with AD and MCI.
Body builders regularly use β-hydroxy β-methylbutyrate (HMB) as a muscle-building supplement to increase exercise-induced gains in muscle size and muscle strength and improve exercise performance. HMB is a very safe supplement, and even after long-term use, it does not exhibit any side effects. Here, we describe that HMB is endowed with a unique property of stimulating hippocampal plasticity. Although HMB is widely used among athletes and body builders as an ergogenic aid, nothing was known about its receptor.
Although the liver is rich in peroxisome proliferator-activated receptor α (PPARα), a nuclear hormone receptor known to participate in fatty acid metabolism,6, we have seen the presence of PPARα in the hippocampus, which is involved in spatial learning and memory via activation of cAMP response element-binding protein (CREB).,,, Here, we found that HMB interacted with the ligand-binding domain of PPARα to activate PPARα and promote hippocampal functions. Accordingly, oral administration of low-dose HMB increased the AMPA- and NMDA-mediated calcium current in hippocampal slices and enhanced memory and learning in 5X familial AD (5XFAD) mice but not 5XFAD mice lacking PPARα (5XFADΔPPARα). Furthermore, HMB treatment lowered plaque load in 5XFAD, but not in 5XFADΔPPARα, mice. These results suggest HMB may be beneficial for patients with AD via PPARα-mediated neuroprotection.

Results

Upregulation of morphological plasticity in hippocampal neurons by HMB

Since the hippocampus, a vital module of memory circuit of the medial temporal lobe, is affected early in AD to display synaptic abnormality, it is believed that upregulation of hippocampal plasticity may be beneficial for AD and other cognitive disorders.,, HMB is a widely used muscle-building supplement, and to understand the effect of HMB on hippocampal plasticity, at first, we examined morphological plasticity. Since dendritic spines are the major sites of excitatory synaptic transmission in the CNS, and accordingly, the functioning of neuronal circuits is influenced by the size and density of dendritic spines,, we monitored the status of dendritic spines. Interestingly, HMB treatment significantly increased the density of spines (Figures 1A–1C ) in primary mouse hippocampal neurons. We further confirmed these observations by quantifying spine size. Similar to the increase in spine density, HMB treatment also augmented spine size in primary hippocampal neurons (Figure 1D). NMDA receptor subunit NR-2A and AMPA-receptor subunit GluR1 are some of the major plasticity-related molecules in the hippocampus. As is evident from immunofluorescence analysis (Figures 1E and 1F), HMB treatment markedly increased the levels of NR2A and GluR1 in primary hippocampal neurons. This was also supported by mean fluorescence intensity (MFI) analysis of NR2A (Figure 1G) and GluR1 (Figure 1H). Several studies have established that calcium influx through NMDA- and AMPA-type glutamate receptors regulates diverse processes including kinase and phosphatase activities, protein trafficking, structural and functional synaptic plasticity, cell growth, cell survival, and apoptosis.,, Therefore, next, we examined whether HMB could arouse the calcium influx in cultured hippocampal neurons. Interestingly, both AMPA (Figure 1I) and NMDA (Figure 1J) elicited a stronger calcium influx following HMB treatment. Since we recorded the NMDA-driven calcium currents in the presence of AMPA-antagonist Naspm (Figure 1I) and AMPA-driven (Figure 1J) calcium currents in the presence of NMDA receptor blocker N20C in HMB-treated primary hippocampal neurons, these results already nullified the contribution of passive calcium currents.
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Figure 1HMB upregulates morphological plasticity in hippocampal neurons
VanGuilder et al. have reported that a decrease in positive clusters for PSD95, an indicator of loss of actual synapses, positively correlates with cognitive decline. Consistent to synaptic degeneration in AD, it has been also shown that the level of synaptosome-associated protein 25 (SNAP25) is significantly lower in AD brains and higher in cerebrospinal fluid (CSF) of subjects with AD. BDNF is probably the most studied neurotrophin from the hippocampus that is known to regulate many of the hippocampus-based biological processes including hippocampal plasticity., On the other hand, being the master regulator of memory and learning, CREB is known to control different plasticity-related molecules like NR-2A, GluR1, PSD95, BDNF, etc., at the transcriptional level., Therefore, we also monitored PSD95, SNAP25, BDNF, and CREB in HMB-treated hippocampal neurons. Interestingly, HMB stimulated the levels of PSD95 (Figures S1A and S1E), SNAP25 (Figures S1B and S1F), BDNF (Figures S1C and S1G), and CREB (Figures S1D and S1H) in hippocampal neurons. These results suggest that HMB is capable of increasing the density of dendritic spines and enhancing the levels of plasticity-related molecules in cultured hippocampal neurons.

Oral administration of HMB upregulates hippocampal functions in 5XFAD mice

Since HMB improves morphological plasticity in cultured hippocampal neurons, next, we examined the effect of HMB in vivo in the hippocampus of 5XFAD mice. At first, we examined whether after oral administration, HMB could enter into the brain. Three days after oral treatment at a dose of 5 mg/kg body weight (wt)/day, HMB was detected in the hippocampus of HMB-fed mice compared with control untreated mice (Figures S2A–S2C), indicating that HMB is capable of crossing the blood-brain barrier. Therefore, after 1 month of HMB treatment via gavage, the ionotropic calcium influx through NMDA and AMPA receptors was monitored in hippocampal slices. As reported earlier,, AMPA- (Figure 2A) and NMDA-dependent (Figure 2B) calcium influx as measured in organotypic hippocampal slices was less in 5XFAD mice compared with age-matched non-transgenic (Tg) mice. However, consistent with the increase in calcium current in cultured hippocampal neurons, oral administration of HMB upregulated AMPA- (Figure 2A) and NMDA-driven (Figure 2B) calcium influx in the hippocampus of 5XFAD mice.
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Figure 2Oral administration of HMB promotes calcium influx in the hippocampus and improves spatial learning and memory of 5XFAD mice
Accordingly, double-label immunofluorescence of hippocampal sections revealed that the level of PSD95 (Figures 2C and 2D) and SNAP25 (Figures S3A and S3B) decreased in the hippocampus of 5XFAD mice compared with non-Tg mice and that HMB feeding upregulated the expression of PSD95 (Figures 2C and 2D) and SNAP25 (Figures S3A and S3B) in the hippocampus of 5XFAD mice. These results were confirmed by western blot analysis of hippocampal extracts with antibodies against PSD95 (Figure 2E) and SNAP25 (Figures S3C) followed by quantification of PSD95 (Figure 2F) and SNAP25 (Figure S3D). As expected, consistent with PSD95 and SNAP25, levels of BDNF (Figures S4A and S4D), total CREB (Figures S4B and S4E), and phosphorylated (phospho)-CREB (Figures S4C and S4F) also decreased in the hippocampus of 5XFAD mice compared with non-Tg mice. However, oral HMB restored/upregulated BDNF (Figures S4A and S4D), total CREB (Figures S4B and S4E), and phospho-CREB (Figures S4C and S4F) in the hippocampus of 5XFAD mice. To confirm these findings further, we performed Golgi staining to visualize the status of dendritic spines in the hippocampus (Figures 2G–2I) by counting pedunculated spines (Figure 2J), non-pedunculated spines (Figure 2K), and total spines (Figure 2L) in the dendritic region of the hippocampal CA1 region. As expected, we found marked loss of pedunculated spines (Figure 2J), non-pedunculated spines (Figure 2K), and total spines (Figure 2L) in the hippocampus of 5XFAD mice compared with non-Tg mice. However, oral HMB treatment restored/improved synaptic connections in the hippocampus of 5XFAD mice as evident from Golgi-stained images (Figure 2I) and the numbers of pedunculated spines (Figure 2J), non-pedunculated spines (Figure 2K), and total spines (Figure 2L).
Next, we investigated the effect of HMB in improving hippocampus-dependent behaviors including memory and learning in 5XFAD mice. The Barnes maze test is used to examine hippocampus-dependent spatial learning and memory., As described before,, 5XFAD mice exhibited diminished spatial behaviors shown by heatmap (Figure 2M), latency (Figure 2N), and errors (Figure 2O) compared with age-matched non-Tg mice. Similarly, 5XFAD mice also performed poorly on T maze, in contrast to non-Tg mice, as demonstrated by positive turn (Figure 2P) and negative turn (Figure 2Q). However, at doses of 5 and 10 mg/kg/day, HMB markedly improved the performance of 5XFAD mice on the Barnes maze (Figure 2M, heatmap; Figure 2N, latency; Figure 2O, errors) and T-maze (Figure 2P, positive turn; Figure 2Q, negative turn). HMB was more effective at a dose of 10 mg/kg/day than 5 mg/kg/day in improving cognitive functions of 5XFAD mice (Figures 2M–2Q).

HMB binds to the ligand-binding domain of PPARα

Next, we wanted to delineate mechanisms by which HMB upregulates morphological plasticity of hippocampal neurons. Although liver is rich in PPARα, a lipid-lowering transcription factor, earlier, we have demonstrated that PPARα is present in the hippocampus and that PPARα also plays an important role in hippocampal plasticity.,,, Therefore, we examined the role of PPARα in this case. Double labeling of hippocampal sections with NeuN and PPARα showed a significant decrease in PPARα in the hippocampus of 5XFAD mice compared with non-Tg mice (Figures S5A and S5B). However, oral HMB treatment markedly upregulated and/or normalized the level of PPARα in the hippocampus of 5XFAD mice (Figures S5A and S5B). On the other hand, we did not see a decrease in PPARβ in the hippocampus of 5XFAD mice compared with non-Tg mice (Figures S5C and S5D), and therefore HMB treatment also did not modulate the level of PPARβ in the hippocampus of 5XFAD mice (Figures S5C and S5D), indicating the specificity of the effect.
Next, we were prompted to investigate the mechanisms of how HMB activates/increases PPARα and whether HMB could assist as a ligand of PPARα. SwissDock, a rigid body protein-ligand docking tool, was employed to explore the interaction between HMB and ligand-binding domain (LBD) of PPARα at a molecular level. According to this analysis, we found that HMB docked in the ligand-binding pocket formed by Ser280, Y314, and H440 (Figures 3A, 3B , S6A, and S6B). To understand the importance of the ligand-binding pocket in the docking of HMB, a key residue of the pocket (Y314) was mutated to D314. When we analyzed the interaction between HMB and Y314D PPARα, HMB was found to be posed far (>5 Å) from the ligand-binding pocket (Figure 3C). This was also reflected by total fitness energy, van der Waal energy, and total free energy (Figure S6C). However, in silico results need to be strengthened by experimental evidence. Therefore, we performed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay in order to validate the interaction between PPARα and HMB. As evident from Figure 3D, HMB indeed exhibited a strong interaction with PPARα. The binding curve resulted in an EC50 value of 3.35 nM with a Hill slope of 0.7710 (Figure 3D). This binding was almost comparable to that of a prototype activator of PPARα (gemfibrozil), which displayed an EC50 value of 4.02 nM with a Hill slope of 0.7949 (Figure 3E).
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Figure 3Characterization of interaction of HMB with PPARα at in silico and molecular levels
To further confirm the interaction between HMB and PPARα, we employed a thermal shift assay (TSA) of PPARα protein with 10 μM HMB. Briefly, full-length PPARα protein (FL-PPARα) was synthesized from HEK293FT cells transduced with lentiviral FL-PPARα. After that, its melting profile was monitored with the help of SYBR green reaction strategy at a range of 27°C–94°C. The typical sigmoidal melting curve clearly showed that our in-house recombinant FL-PPARα protein is conformationally stable (Figure 3F). Our TSA also revealed that 10 μM HMB strongly shifted the melting curve of FL-PPARα by 7°C (Figure 3F). To confirm in silico results further, we also performed a TSA with Y314DPPARα protein, which showed that 10 μM HMB could shift the melting curve of Y314DPPARα by only 0.76°C (Figure 3G), clearly indicating that HMB binds to the ligand-binding pocket of PPARα.
To confirm the functional significance of this finding, primary astrocytes isolated from PPARα−/− mice were transduced with lenti-FL-PPARα and lenti-Y314D-PPARα followed by HMB treatment. Consistent with structural and biophysical analyses, HMB treatment upregulated PPARα in PPARα−/− astrocytes (Figures 3H and 3I) that were transduced with lenti-FL-PPARα, but not lenti-Y314D-PPARα, further highlighting the importance of the interaction of HMB with the PPARα LBD in the activation of PPARα.

HMB-mediated upregulation of structural plasticity is dependent on its interaction with Y314 residue of PPARα

Next, we examined whether HMB augmented synaptic function via PPARα. Quantification of dendritic spine density is an important measure to evaluate hippocampal functions. Therefore, we employed a phalloidin-based quantification analysis of dendritic spines in HMB-treated hippocampal neurons. HMB increased spine density in wild-type (WT) (Figures 1A–1D), but not PPARα−/− (Figures 4A–4C ), hippocampal neurons. Next, PPARα−/− hippocampal neurons were transduced with lenti-FL-PPARα for 2 days followed by overnight treatment with HMB. Interestingly, introduction of FL-PPARα significantly increased spine density in HMB-stimulated hippocampal neurons (Figures 4A and 4B). We further confirmed these observations by measuring spine size (Figure 4C) under the different treatment conditions. These results suggest that HMB upregulates morphological plasticity in hippocampal neurons via PPARα.
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Figure 4HMB stimulates structural plasticity in hippocampal neurons via PPARα
Along with the estimation of dendritic spine density, the measurement of calcium influx through ionotropic receptors including NMDA and AMPA receptors is considered another reliable procedure to evaluate synaptic function.,, Interestingly, HMB could not induce AMPA- and NMDA-driven calcium influx in cultured hippocampal neurons isolated from PPARα−/− mice (Figures 4D and 4E) compared with WT mice (Figures 1I and 1J), suggesting that HMB involves PPARα to upregulate calcium influx in hippocampal neurons. Next, to delineate a direct role of the Y314 residue of PPARα in HMB-induced calcium influx, PPARα−/− hippocampal neurons were transduced with lenti-FL-PPARα and lenti-Y314DPPARα for 2 days followed by stimulation with 10 μM HMB. Remarkably, HMB increased both AMPA- and NMDA-mediated calcium currents in lenti-FL-PPARα-transduced (Figures 4F and 4G), but not lenti-Y314DPPARα-transduced (Figures 4H and 4I), PPARα−/− hippocampal neurons. These results suggest that the binding of HMB with the Y314 residue of the PPARα LBD is important for HMB-mediated upregulation of calcium influx in hippocampal neurons through NMDA- and AMPA-sensitive receptors.

HMB increases structural plasticity and protects memory and learning in 5XFAD mice via PPARα

Next, we investigated whether HMB required PPARα to protect hippocampal functions in vivo in mouse brain. Therefore, we used 5XFADΔPPARα mice (5XFAD mice lacking PPARα)., Seven-month-old 5XFADΔPPARα mice (n = 6) were fed with HMB for 30 days followed by monitoring of the ionotropic calcium influx through NMDA and AMPA receptors in hippocampal slices. In contrast to the upregulation of AMPA- and NMDA-dependent calcium influx in organotypic hippocampal slices of 5XFAD mice by HMB (Figures 2A and 2B), this supplement remained unable to stimulate calcium influx in hippocampal slices of 5XFADΔPPARα mice (Figures 5A and 5B ). Although HMB treatment upregulated PSD95 in the hippocampus of 5XFAD mice (Figures 2C–2F), an increase in PSD95 protein was not found in the hippocampus of HMB-treated 5XFADΔPPARα mice (Figures 5C–5F). Accordingly, oral HMB increased the level of SNAP25 in the hippocampus of 5XFAD mice (Figures S3A–S3D) but not of 5XFADΔPPARα mice (Figures S7A–S7D). Similarly, HMB treatment also remained unable to increase the level of BDNF (Figures S8A and S8D), CREB (Figures S8B and S8E), and phospho-CREB (Figures S8C and S8F) in the hippocampus of 5XFADΔPPARα mice. These results suggest that HMB requires PPARα in upregulating morphological plasticity in vivo in the hippocampus of 5XFAD mice.
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Figure 5Oral HMB does not improve spatial learning and memory of 5XFAD mice lacking PPARα
Next, we explored the role of HMB in educating 5XFADΔPPARα mice in hippocampus-dependent behaviors including memory and learning. Although HMB treatment increased the performance of 5XFAD mice on the Barnes maze (Figures 2L–2N) and T maze (Figures 2O–2P), this supplement could not protect spatial learning and memory in 5XFADΔPPARα mice, as evidenced from the heatmap (Figure 5G), latency (Figure 5H), and error (Figure 5I) from the Barnes maze and positive turn (Figure 5J) and negative turn (Figure 5K) from the T maze. These results demonstrate that HMB improves memory and learning in 5XFAD mice via PPARα.

Oral HMB lowers the plaque burden in the brain of 5XFAD mice

Since amyloid plaques play an important role in the disease process of AD and such pathology is widespread in 5XFAD mice,,,,, we also examined whether oral administration of HMB was capable of decreasing the amyloid load in the hippocampus, the most affected brain region in AD, of 5XFAD mice. Aβ peptides are the main component of the amyloid plaques, and both common isoforms Aβ40 and Aβ42 are recognized by 6E10 monoclonal antibodies (mAbs). DAB immunostaining with 6E10 mAbs showed a remarkable increase in the Aβ in the hippocampus and cortex of 5XFAD mice compared with non-Tg mice (Figures 6A–6C ). Quantification of plaques in the hippocampus (Figures 6D–6F) and cortex (Figures 6G–6I) also corroborated the increase in plaques in the brain of 5XFAD mice compared with non-Tg mice. However, oral administration of HMB significantly decreased the level of Aβ in the hippocampus and cortex of 5XFAD mice (Figures 6A–6I). Immunoblot analysis of hippocampal homogenates with 6E10 mAbs also demonstrated a markedly higher level of Aβ peptides in the CNS of 5XFAD mice compared with non-Tg mice (Figures 6J and 6K). However, similar to DAB staining, treatment of 5XFAD mice with HMB led to a significant decrease in Aβ (Figures 6J and 6K).
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Figure 6HMB treatment reduces plaque burden in the hippocampus and cortex of 5XFAD mouse model of AD
To further confirm the deposition of amyloid plaques in the brain, we performed double labeling of hippocampal sections with thioflavin-S (thio-S), a classic amyloid-binding dye for the detection of the β-pleated sheet of the amyloid plaques, and 6E10. Consistent with the DAB and western blot results, a marked abundance of thio-S-positive and Aβ-immunoreactive plaques were observed in the CNS of 5XFAD mice (Figures 7A and S9A). However, treatment of 5XFAD mice with HMB decreased the plaque load (Figure 7A). Quantitative analysis of thio-S staining also showed that HMB treatment led to a significant decline in thio-S-positive area (Figure 7B), thio-S puncta (Figure 7C), and thio-S puncta size (Figure 7D) in the hippocampus of 5XFAD mice. Moreover, ELISAs indicated an increase in Aβ1-42 (Figure 7E) and Aβ1-40 (Figure 7F) in serum of 5XFAD mice compared with non-Tg mice. ELISAs of TBS-extracted (Figures S9B and S9C) and (TBS+Triton X-100)-extracted (Figures S9D and S9E) hippocampal extracts also showed upregulation of Aβ1-40 (Figures S9B and S9D) and Aβ1-42 (Figures S9C and S9E) in 5XFAD mice compared with non-Tg mice. However, consistent with the decrease in amyloid pathology in the CNS, HMB treatment decreased the level of both Aβ1-42 (Figures 7E, S9C, and S9E) and Aβ1-40 (Figures 7F, S9B, and S9D) in serum (Figures 7E and 7F) and the hippocampus (Figures S9B–S9E) of 5XFAD mice.
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Figure 7HMB requires PPARα to decrease Aβ plaque in the brain of 5XFAD mice

Oral administration of HMB reduces plaques from the hippocampus of 5XFAD mice via PPARα

Since HMB protects memory and learning in 5XFAD mice via PPARα, next, we examined whether HMB also required PPARα to lower plaques from the hippocampus of 5XFAD mice. Although HMB treatment decreased plaques from the brain of 5XFAD mice (Figure 6), this supplement remained unable to reduce amyloid plaques from the hippocampus and cortex of 5XFADΔPPARα mice as is evident from DAB immunostaining of hippocampal and cortical sections of 5XFADΔPPARα mice (Figures S10A–S10C). Quantification of plaques in the hippocampus (Figures S10D–S10F) and cortex (Figures S10G–S10I) also showed that HMB treatment remained unable to decrease the number (Figures S10D and S10G), area (Figures S10E and S10H), and density (Figures S10F and S1I) of plaques in 5XFADΔPPARα mice. These results were also corroborated by western blot analysis of hippocampal extracts (Figures S10J–S10K). Thio-S and 6E10 double labeling also showed that HMB could not inhibit the level of amyloid plaques (Figures 7A and S9A), decrease thio-S area (Figure 7B), reduce thio-S puncta (Figure 7C), or lower thio-S puncta size (Figure 7D) in the hippocampus of 5XFADΔPPARα mice. Consequently, HMB treatment also could not decrease Aβ1-42 (Figures 7E, S9C, and S9E) or Aβ1-40 (Figures 7F, S9B, and S9D) in serum (Figures 7E and 7F) and the hippocampus (Figures S9B–S9E) of 5XFADΔPPARα mice. Together, these results also suggest that HMB is unable to decrease plaques from the brain of 5XFAD mice in the absence of PPARα.

Discussion

At present, no effective treatment is available to prevent or halt the progression of AD. Therefore, describing non-toxic molecules for refining hippocampal functions, halting cognitive decline (the central clinical symptom of AD), and lowering senile plaques (one of the pathological markers of AD) is an important area of research. HMB is a widely used body-building supplement among muscle builders and combat sports athletes. Here, we describe that oral HMB is capable of improving hippocampal plasticity, restoring cognitive functions, and reducing plaque load in 5XFAD mouse models of AD. Since HMB is a non-toxic and easily available supplement, these results suggest that oral HMB may be used as a therapeutic supplement in patients with AD and MCI.
The hippocampus is endowed with unique functions of processing, organizing, and storing memories.,
Therefore, upregulation of hippocampal plasticity is an important area of research for better therapeutic outcome in patients with AD. CREB is considered the master regulator of memory and learning, as almost all molecules, including BDNF, involved in hippocampal plasticity are transcriptionally controlled by CREB.,, On the other hand, PPARα is a lipid-lowering transcription factor that, being abundant in the liver, helps in the reduction of triglycerides and free fatty acids via stimulation of peroxisomal β-oxidation of very-long-chain fatty acids.,, Recently, we have seen that PPARα is also present in different regions of the brain including the hippocampus.,, Interestingly, earlier, we demonstrated that the level of CREB is lower in the hippocampus of PPARα−/− mice, that CREB is transcriptionally regulated by PPARα, and that activation of PPARα stimulates hippocampal plasticity via an increase in CREB. Moreover, upregulation of CREB and rebuilding of spatial learning and memory in PPARα−/− mice by lentiviral transfer of PPARα into the hippocampus proposes an important role of PPARα in cognitive functions. Here, we have also seen that HMB treatment increases the level of CREB and CREB-associated plasticity-related molecules in the hippocampus, stimulates calcium oscillation in hippocampal slices, and improves spatial learning and memory in 5XFAD, but not 5XFADΔPPARα, mice. These results suggest that oral HMB is capable of upregulating CREB and improving CREB-dependent hippocampal functions in 5XFAD mice via PPARα.
Many strategies for the development of novel therapeutics for AD have been focused on targeting the senile plaques that are formed by abnormal deposition of Aβ. Senile plaques are broadly classified into two categories, such as diffuse and dense-core plaques. Diffuse plaques are thio-S negative, non-neuritic, and frequently observed in aged people who are cognitively intact. On the other hand, dense-core plaques that are present in the brains of patients with clinically identified AD stain positively for thio-S and are composed of fibrillar Aβ. Mechanisms by which cerebral plaque level could be reduced are poorly understood. While the upregulation of the ADAM10-mediated nonamyloidogenic pathway inhibits the formation of amyloid plaques in neurons, stimulation of the TFEB-driven lysosome-autophagy pathway increases the degradation of amyloid plaques. On one hand, activation of PPARα stimulates the nonamyloidogenic pathway via transcriptional upregulation of ADAM10. On the other, activated PPARα also leads to an increase in lysosomal biogenesis and autophagy via transcriptional stimulation of TFEB., It has been demonstrated that PPARα, but neither PPARβ nor PPARγ, is directly recruited to the promoters of ADAM10 and TFEB genes in response to gemfibrozil treatment. Therefore, PPARα plays a central role in controlling the level of plaques in the brain. Astrocytes are the major cell type in the brain, and recently, we also described that activation of PPARα is capable of enhancing astroglial uptake and degradation of Aβ. It is important to mention that HMB is also capable of activating PPARα in primary astrocytes via interaction with the Y314 residue of PPARα (Figures 3H and 3I). Our current finding of lowering amyloid plaques by HMB treatment in 5XFAD, but not 5XFADΔPPARα (5XFAD mice lacking PPARα), mice suggests that, similar to the improvement in cognitive functions, HMB treatment lowers plaque load in 5XFAD mice via PPARα.
How does HMB involve PPARα to exhibit its memory-boosting and plaque-lowering activities? It is not known whether HMB is a ligand of PPARα. However, in cultured cells, HMB induces the activation of PPARα, as evidenced by increased nuclear translocation. The LBD of PPARα is quite large, with a 1,400-Å-wide pocket size that allows lipophilic compounds such as medium- and long-chain fatty acids to be docked inside. However, a small polar environment is also maintained within the PPARα LBD by a catalytic triad of Ser280, Tyr314, and His440 to ultimately allow small polar compounds to be docked inside. It is believed that these three key residues stabilize the docking of partially polar compounds via the formation of H-bonds. HMB is a negatively charged polar compound, and according to our in silico analysis, it forms H-bonds with the catalytic triad of the PPARα LBD. Upon analysis of the interaction of HMB with PPARα by different biophysical approaches such as TR-FRET and protein TSA, we have also seen strong binding of HMB with the PPARα LBD. Accordingly, HMB failed to activate PPARα, could not enhance morphological plasticity of hippocampal neurons, and remained unable to stimulate AMPA- and NMDA-induced calcium influx in mutated (Y314D) PPARα-transduced PPARα−/− hippocampal neurons, underlining the functional significance of HMB’s interaction with the Y314 residue of PPARα LBD.
HMB is a very safe supplement, and even after long-term use, it does not exhibit any side effects. For muscle building, HMB is recommended at a dose of 3 g per day per adult. However, this 3 g should not be taken in one serving but rather split into 3 servings throughout the day, making it around 1g per serving per adult. If our mouse dose of HMB (5 or 10 mg/kg body wt/day) is translated to human, HMB at a dose of 400 or 800 mg per adult per day may be beneficial to control AD-related symptoms and pathology. Therefore, the dose at which HMB may improve memory and learning and lower the plaque burden in patients with AD is much lower than the dose that is being used to support body building in humans. In a 12 week, randomized, double-blind, placebo-controlled crossover study among 42 highly trained combat sports athletes, HMB treatment led to increase in fat-free mass with simultaneous decrease in fat mass. HMB treatment also increased aerobic and anaerobic capacity among combat sports athletes. Therefore, oral HMB should not exhibit toxicity in patients with AD and MCI.
Myokines/hepatokines, peptides produced and released by muscle/liver, are known to mediate communication between muscle and other organs. Recent studies have shown that these molecules may play a role in streptozotocin-induced neuronal damage. Since HMB is a muscle-building drug, future studies may be directed at defining the mechanism behind muscle-to-brain crosstalk and whether myokines/hepatokines play a role in HMB-mediated neuroprotection. In summary, HMB, a commonly used body-building supplement in human, binds to the LBD of PPARα to stimulate CREB and promote hippocampal plasticity via PPARα. After oral administration, HMB stimulates hippocampal function, defends spatial learning and memory, and lowers cerebral plaque load in an animal model of AD via PPARα. Therefore, HMB supplement may be beneficial for AD as well as other cognitive disorders.

Limitations of the study

Here, we have described that oral HMB reduces plaques and improves cognitive functions in 5XFAD mouse models of AD. Being a muscle-building supplement, HMB is known to strengthen muscle, and this property of HMB may contribute to HMB-mediated improved performance on the Barnes maze and T maze. However, here, we do not know whether this classical muscle-building property of HMB has any role in improved maze performance of 5XFAD mice. Therefore, experiments may be planned in the future to address these issues.
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